Compositions and methods for inhibiting expression of the HAMP gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the HAMP gene (HAMP gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the HAMP gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by HAMP gene expression and the expression of the HAMP gene using the pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. application Ser. No. 13/590,783,filed Aug. 21, 2012 (now U.S. Pat. No. 8,470,799); U.S. application Ser.No. 13/184,087, filed Jul. 15, 2011 (now U.S. Pat. No. 8,268,799); U.S.application Ser. No. 12/757,497, filed Apr. 9, 2010 (now U.S. Pat. No.8,163,711); U.S. application Ser. No. 11/859,288, filed Sep. 21, 2007(abandoned); U.S. Provisional Application No. 60/846,266, filed Sep. 21,2006; and U.S. Provisional Application No. 60/870,253, filed Dec. 15,2006. The entire contents of these applications are hereby incorporatedby reference in the present application.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theHAMP gene and the use of the dsRNA to treat pathological processes whichcan be mediated by down regulating HAMP, such as anemia and otherdiseases associated with lowered iron levels.

BACKGROUND OF THE INVENTION

The discovery of the hepcidin peptide and characterization of its gene,HAMP,⁴ has led to the revision of previous models for the regulation ofiron homeostasis and the realisation that the liver plays a key role indetermining iron absorption from the gut and iron release from recyclingand storage sites. Perhaps the most striking example has been to changethe pathogenic model of HFE-related hereditary haemochromatosis from thecrypt-programming model centered on the duodenal absorptive enterocyteto the hepcidin model centered on the hepatocyte.^(5,6) In summary, thehepcidin model proposes that the rate of iron efflux into the plasmadepends primarily on the plasma level of hepcidin; when iron levels arehigh the synthesis of hepcidin increases and the release of iron fromenterocytes and macrophages is diminished. Conversely when iron storesdrop, the synthesis of hepcidin is down-regulated and these cellsrelease more iron.

In order to describe the postulated major role of hepcidin it isnecessary to understand the function of ferroportin, a protein firstcharacterised in 2000. Ferroportin is the major iron export proteinlocated on the cell surface of enterocytes, macrophages and hepatocytes,the main cells capable of releasing iron into plasma for transport bytransferrin.⁷

The major iron recycling pathway is centered on the degradation ofsenescent red cells by reticuloendothelial macrophages located in bonemarrow, hepatic Kupffer cells and spleen. The exit of iron from thesemacrophages is controlled by ferroportin. The role of the hepatocyte iscentral to the action of ferroportin, because the hepatocyte is proposedto sense body iron status and either release or down-regulate hepcidin,which then interacts with ferroportin to modulate the release ofcellular iron. Hepcidin directly binds to ferroportin and decreases itsfunctional activity by causing it to be internalized from the cellsurface and degraded.⁸

Increased hepcidin synthesis is thought to mediate iron metabolism intwo clinically important circumstances, shown schematically in FIG. 1.In individuals who do not harbour mutations causing haemochromatosis,the hepatocyte is thought to react to either an increase in ironsaturation of transferrin or to increased iron stores in hepatocytesthemselves, by inducing the synthesis of hepcidin by an as yet unknownmechanism. Thus the physiological response to iron overload under normalcircumstances would be the hepcidin mediated shut down of ironabsorption (enterocyte), recycling (macrophage) and storage(hepatocyte).

The synthesis and release of hepcidin is also rapidly mediated bybacterial lipopolysaccaride and cytokine release, especiallyinterleukin-6 Thus the hepcidin gene is an acute-phase responsive genewhich is overexpressed in response to inflammation. Cytokine mediatedinduction of hepcidin caused by inflammation or infection is now thoughtto be responsible for the anaemia of chronic disease, where iron isretained by the key cells that normally provide it, namely enterocytes,macrophages and hepatocytes. Retention of iron leads to the hallmarkfeatures of the anaemia of chronic disease, low transferrin saturation,iron-restricted erythropoeisis and mild to moderate anaemia.⁹ The natureof the hepcidin receptor is presently unknown, however an excitingfuture prospect may be the development of agents to block the receptorwith the aim of treating the anaemia of chronic disease, a common oftenintractable clinical problem.

Down-regulation of hepcidin synthesis results in increased iron release,which arises in the two situations shown schematically in FIG. 2. Themain causes of non-HFE haemochromatosis are mutations in eitherferroportin, transferrin receptor 2, hepcidin or hemojuvelin genes.Classical HFE haemochromatosis, and all types of non-HFEhaemochromatosis thus far studied with the exception of ferroportinrelated haemochromatosis, are characterised by inappropriate hepcidindeficiency. In these circumstances, hepatocytes become iron loaded,because their uptake of transferrin bound iron from the circulation isassumed to exceed that of ferroportin mediated export. Hepcidindeficiency causes increased ferroportin mediated iron export, resultingin increased enterocyte absorption of iron and perhaps quantitativelymore important, enhanced export of recycled iron onto plasma transferrinby macrophages. Hepcidin is also suppressed in thalassaemic syndromes,both β thalassaemia major and intermedia and congenital dyserythropoeticanaemic type 1, where iron absorption is inappropriately stimulateddespite the presence of massive iron overload.¹⁰

As shown in FIG. 2, anaemia and hypoxia both trigger a decrease inhepcidin levels. These discoveries were made in animal models and needto be further studied to show they are applicable in humans. Two animalmodels of anaemia in mice were used to demonstrate a dramatic decreasein hepcidin synthesis where anaemia was provoked either by excessivebleeding or haemolysis.¹¹ This is postulated to permit the rapidmobilisation of iron from macrophages and enterocytes necessary to allowfor the increased erythropoietic activity triggered by erythropoietinrelease. The same study showed down-regulation of hepcidin synthesis canbe triggered by hypoxia alone, and mice housed in hypobaric hypoxiachambers simulating an altitude of 5,500 m also showed a rapid decreasein hepcidin.

In summary, hepcidin provides a unifying hypothesis to explain thebehaviour of iron in two diverse but common clinical conditions, theanaemia of chronic disease and both HFE and non-HFE haemochromatosis.The pathophysiology of hepcidin has been sufficiently elucidated tooffer promise of therapeutic intervention in both of these situations.Administering either hepcidin or an agonist could treathaemochromatosis, where the secretion of hepcidin is abnormally low.

1. Park C H, Valore E V, Waring A J, Ganz T. Hepcidin, a urinaryantimicrobial peptide synthesized in the liver. J Biol. Chem. 2001;276:7806-10.

2. Pigeon C, Ilyin G, Courselaud B, et al. A new mouse liver-specificgene, encoding a protein homologous to human antimicrobial peptidehepcidin, is overexpressed during iron overload. J Biol. Chem. 2001;276:7811-9.

3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator ofanemia of inflammation. Blood. 2003; 102:783-8.

4. Roetto A, Papanikolaou G, Politou M, et al. Mutant antimicrobialpeptide hepcidin is associated with severe juvenile hemochromatosis.Nat. Genet. 2003; 33:21-2.

5. Fleming R E. Advances in understanding the molecular basis for theregulation of dietary iron absorption. Curr Opin Gastroenterol. 2005;21:201-6.

6. Pietrangelo A. Hereditary hemochromatosis—a new look at an olddisease. N Engl J. Med. 2004; 350:2383-97.

7 Donovan A, Brownlie A, Zhou Y, et al. Positional cloning of zebrafishferroportinl identifies a conserved vertebrate iron exporter. Nature.2000; 403:776-81.

8. Nemeth E, Tuttle M S, Powelson J, et al. Hepcidin regulates cellulariron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306:2090-3.

9. Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J. Med.2005; 352:1011-23.

10 Papanikolaou G, Tzilianos M, Christakis J I, et al. Hepcidin in ironoverload disorders. Blood. 2005; 105:4103-5.

11 Nicolas G, Chauvet C, Viatte L, et al. The gene encoding the ironregulatory peptide hepcidin is regulated by anemia, hypoxia, andinflammation. J Clin Invest. 2002; 110:1037-44.

The anemia of inflammation, commonly observed in patients with chronicinfections, malignancy, trauma, and inflammatory disorders, is awell-known clinical entity. Until recently, little was understood aboutits pathogenesis. It now appears that the inflammatory cytokine IL-6induces production of hepcidin, an iron-regulatory hormone that may beresponsible for most or all of the features of this disorder. (Andrews NC. J Clin Invest. 2004 May 1; 113(9): 1251-1253). As such, downregulation of hepcidin in anemic patients will lead to a reduction ininflammation associated with such anemia.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pathological processes which can be mediated by downregulating HAMP gene expression, there remains a need for agents thatcan inhibit HAMP gene expression and that can treat diseases associatedwith HAMP gene expression such as anemia and other diseases associatedwith lowered iron levels.

SUMMARY OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by down regulating the proprotein hepcidingene/protein (HAMP) by using double-stranded ribonucleic acid (dsRNA) tosilence HAMP expression.

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the HAMPgene in a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions that canmodulated by down regulating the expression of the HAMP gene, such asanemia and other diseases associated with lowered iron levels. The dsRNAof the invention comprises an RNA strand (the antisense strand) having aregion which is less than 30 nucleotides in length, generally 19-24nucleotides in length, and is substantially complementary to at leastpart of an mRNA transcript of the HAMP gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the HAMP gene.The dsRNA comprises at least two sequences that are complementary toeach other. The dsRNA comprises a sense strand comprising a firstsequence and an antisense strand comprising a second sequence. Theantisense strand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding HAMP, and the regionof complementarity is less than 30 nucleotides in length, generally19-24 nucleotides in length. The dsRNA, upon contacting with a cellexpressing the HAMP, inhibits the expression of the HAMP gene by atleast 40%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Tables 1 or 3 and the second sequence isselected from the group consisting of the antisense sequences of Tables1 or 3. The dsRNA molecules of the invention can be comprised ofnaturally occurring nucleotides or can be comprised of at least onemodified nucleotide, such as a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative. Alternatively, themodified nucleotide may be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such modifiedsequence will be based on a first sequence of said dsRNA selected fromthe group consisting of the sense sequences of Tables 1 or 3 and asecond sequence selected from the group consisting of the antisensesequences of Tables 1 or 3.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the HAMP gene in anorganism, generally a human subject, comprising one or more of the dsRNAof the invention and a pharmaceutically acceptable carrier or deliveryvehicle. Preferable the carrier or delivery vehicle will be one thatselectively targets the siRNA to the liver.

In another embodiment, the invention provides a method for inhibitingthe expression of the HAMP gene in a cell, comprising the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding HAMP, and wherein the        region of complementarity is less than 30 nucleotides in length,        generally 19-24 nucleotides in length, and wherein the dsRNA,        upon contact with a cell expressing the HAMP, inhibits        expression of the HAMP gene by at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        HAMP gene, thereby inhibiting expression of the HAMP gene in the        cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes which can be mediated bydown regulating HAMP gene expression, e.g. anemia and other diseasesassociated with lowered iron levels, comprising administering to apatient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe dsRNAs of the invention.

In another embodiment, the invention provides vectors for inhibiting theexpression of the HAMP gene in a cell, comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of the HAMP gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating induction of liver hepcidinsynthesis, which decreases iron export from absorptive cells(enterocytes), recycling cells (macrophages) and storage cells(hepatocytes).

FIG. 2 is a schematic diagram illustrating that down-regulation of liverhepcidin synthesis increases iron export from absorptive cells(enterocytes), recycling cells (macrophages) and storage cells(hepatocytes). The box labelled ‘FIFE- and Non-HFE haemochromatosis.(not FP disease)’ refers to HFE- and non-HFE haemochromatosis with thesole exception of ferroportin disease.

FIG. 3 is a graph showing the silencing activity of humanhepcidin-siRNAs.

FIG. 4 is a graph showing the silencing activity of humanhepcidin-siRNAs.

FIG. 5 is a graph showing the silencing activity of humanhepcidin-siRNAs.

FIG. 6 is a graph showing the activity of mouse hepcidin siRNAs.

FIG. 7 shows the structure of the ND-98 lipid used in generatingliposomes used for in vivo studies.

FIGS. 8A and 8B are graphs of in vivo activity of a liposomal formulatedmouse hepcidin siRNA.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by the down regulation of the HAMP gene, by usingdouble-stranded ribonucleic acid (dsRNA) to silence the HAMP gene thusproviding treatment for diseases such as anemia and other diseasesassociated with lowered iron levels.

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the HAMPgene in a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases that can be modulated by down regulating the expression of theHAMP gene. dsRNA directs the sequence-specific degradation of mRNAthrough a process known as RNA interference (RNAi).

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of the HAMP gene.The use of these dsRNAs enables the targeted degradation of an mRNA thatis involved in sodium transport. Using cell-based and animal assays, thepresent inventors have demonstrated that very low dosages of these dsRNAcan specifically and efficiently mediate RNAi, resulting in significantinhibition of expression of the HAMP gene. Thus, the methods andcompositions of the invention comprising these dsRNAs are useful fortreating pathological processes which can be mediated by down regulatingHAMP, such as in the treatment of anemia and other diseases associatedwith lowered iron levels.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of thetarget HAMP gene, as well as compositions and methods for treatingdiseases that can be modulated by down regulating the expression ofHAMP, such as anemia and other diseases associated with lowered ironlevels. The pharmaceutical compositions of the invention comprise adsRNA having an antisense strand comprising a region of complementaritywhich is less than 30 nucleotides in length, generally 19-24 nucleotidesin length, and is substantially complementary to at least part of an RNAtranscript of the HAMP gene, together with a pharmaceutically acceptablecarrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the HAMP gene, and methods of using thepharmaceutical compositions to treat diseases that can be modulated bydown regulating the expression of HAMP.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “HAMP” refers to the hepcidin gene or protein (alsoknown as LEAP). mRNA sequences to HAMP are provided as human: Genbankaccession NM_(—)021175.2.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the HAMP gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding HAMP). For example, a polynucleotide iscomplementary to at least a part of a HAMP mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding HAMP.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where separate RNA molecules, such dsRNAare often referred to in the literature as siRNA (“short interferingRNA”). Where the two strands are part of one larger molecule, andtherefore are connected by an uninterrupted chain of nucleotides betweenthe 3′-end of one strand and the 5′ end of the respective other strandforming the duplex structure, the connecting RNA chain is referred to asa “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strandsare connected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.In addition, as used in this specification, “dsRNA” may include chemicalmodifications to ribonucleotides, including substantial modifications atmultiple nucleotides and including all types of modifications disclosedherein or known in the art. Any such modifications, as used in an siRNAtype molecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. For clarity, chemical caps or non-nucleotidechemical moieties conjugated to the 3′ end or 5′ end of an siRNA are notconsidered in determining whether an siRNA has an overhang or is bluntended.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to the HAMP gene, herein refer to the at least partial suppressionof the expression of the HAMP gene, as manifested by a reduction of theamount of mRNA transcribed from the HAMP gene which may be isolated froma first cell or group of cells in which the HAMP gene is transcribed andwhich has or have been treated such that the expression of the HAMP geneis inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{20mu}{in}\mspace{14mu}{control}\mspace{20mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to HAMP genetranscription, e.g. the amount of protein encoded by the HAMP gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g. apoptosis. In principle, HAMP gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of the HAMP gene by a certain degree and therefore isencompassed by the instant invention, the assay provided in the Examplesbelow shall serve as such reference.

For example, in certain instances, expression of the HAMP gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention. In someembodiments, the HAMP gene is suppressed by at least about 60%, 70%, or80% by administration of the double-stranded oligonucleotide of theinvention. In some embodiments, the HAMP gene is suppressed by at leastabout 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. Table 2 provides a wide range ofvalues for inhibition of expression obtained in an in vitro assay usingvarious HAMP dsRNA molecules at various concentrations.

As used herein in the context of HAMP expression, the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes which can be mediated by down regulating the HAMPgene. In the context of the present invention insofar as it relates toany of the other conditions recited herein below (other thanpathological processes which can be mediated by down regulating the HAMPgene), the terms “treat”, “treatment”, and the like mean to relieve oralleviate at least one symptom associated with such condition, or toslow or reverse the progression of such condition. For example, in thecontext of anemia and other diseases associated with lowered ironlevels, treatment will involve an increase in serum iron levels. Examplepatient populations that can benefit from such a treatment include, butare not limited to, individuals having anemia as a result of chronicrenal failure, cancer patients, patients with chronic inflammatorydisease as well as patients with chronic GI bleeding, such as withchronic ulcers or colon tumors.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes which can be mediated by down regulating the HAMPgene on or an overt symptom of pathological processes which can bemediated by down regulating the HAMP gene. The specific amount that istherapeutically effective can be readily determined by ordinary medicalpractitioner, and may vary depending on factors known in the art, suchas, e.g. the type of pathological processes which can be mediated bydown regulating the HAMP gene, the patient's history and age, the stageof pathological processes which can be mediated by down regulating HAMPgene expression, and the administration of other anti-pathologicalprocesses which can be mediated by down regulating HAMP gene expression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof and are described in more detailbelow. The term specifically excludes cell culture medium.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the HAMP gene ina cell or mammal, wherein the dsRNA comprises an antisense strandcomprising a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of the HAMP gene, andwherein the region of complementarity is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and wherein said dsRNA,upon contact with a cell expressing said HAMP gene, inhibits theexpression of said HAMP gene by at least 40%. The dsRNA comprises twoRNA strands that are sufficiently complementary to hybridize to form aduplex structure. One strand of the dsRNA (the antisense strand)comprises a region of complementarity that is substantiallycomplementary, and generally fully complementary, to a target sequence,derived from the sequence of an mRNA formed during the expression of theHAMP gene, the other strand (the sense strand) comprises a region whichis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30, moregenerally between 18 and 25, yet more generally between 19 and 24, andmost generally between 19 and 21 base pairs in length. Similarly, theregion of complementarity to the target sequence is between 15 and 30,more generally between 18 and 25, yet more generally between 19 and 24,and most generally between 19 and 21 nucleotides in length. The dsRNA ofthe invention may further comprise one or more single-strandednucleotide overhang(s). The dsRNA can be synthesized by standard methodsknown in the art as further discussed below, e.g., by use of anautomated DNA synthesizer, such as are commercially available from, forexample, Biosearch, Applied Biosystems, Inc. In a preferred embodiment,the HAMP gene is the human HAMP gene. In specific embodiments, theantisense strand of the dsRNA comprises a strand selected from the sensesequences of Tables 1 or 3 and a second sequence selected from the groupconsisting of the antisense sequences of Tables 1 or 3. Alternativeantisense agents that target elsewhere in the target sequence providedin Tables 1 or 3 can readily be determined using the target sequence andthe flanking HAMP sequence.

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided in Tables 1 or3. In other embodiments, the dsRNA comprises at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofan mRNA generated in the expression of the HAMP gene. Generally, thedsRNA comprises two oligonucleotides, wherein one oligonucleotide isdescribed as the sense strand in Tables 1 or 3 and the secondoligonucleotide is described as the antisense strand in Tables 1 or 3

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1 or 3, the dsRNAs of theinvention can comprise at least one strand of a length of minimally 21nt. It can be reasonably expected that shorter dsRNAs comprising one ofthe sequences of Tables 1 or 3 minus only a few nucleotides on one orboth ends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs comprising a partial sequence of at least 15, 16,17, 18, 19, 20, or more contiguous nucleotides from one of the sequencesof Tables 1 or 3, and differing in their ability to inhibit theexpression of the HAMP gene in a FACS assay as described herein below bynot more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the invention. FurtherdsRNAs that cleave within the target sequence provided in Tables 1 or 3can readily be made using the HAMP sequence and the target sequenceprovided.

In addition, the RNAi agents provided in Table 1 identify a site in theHAMP mRNA that is susceptible to RNAi based cleavage. As such thepresent invention further includes RNAi agents that target within thesequence targeted by one of the agents of the present invention. As usedherein a second RNAi agent is said to target within the sequence of afirst RNAi agent if the second RNAi agent cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thefirst RNAi agent. Such a second agent will generally consist of at least15 contiguous nucleotides from one of the sequences provided in Table 1coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in the HAMP gene. For example, thelast 15 nucleotides of SEQ ID NO:1 (minus the added AA sequences)combined with the next 6 nucleotides from the target HAMP gene producesa single strand agent of 21 nucleotides that is based on one of thesequences provided in Table 1.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the HAMP gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the HAMP gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the HAMP gene is important,especially if the particular region of complementarity in the HAMP geneis known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Chemical modifications may include,but are not limited to 2′ modifications, modifications at other sites ofthe sugar or base of an oligonucleotide, introduction of non-naturalbases into the oligonucleotide chain, covalent attachment to a ligand orchemical moiety, and replacement of internucleotide phosphate linkageswith alternate linkages such as thiophosphates. More than one suchmodification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough use of purine analogues. Generally, the chemical groups that canbe used to modify the dsRNA include, without limitation, methylene blue;bifunctional groups, generally bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone embodiment, the linker is a hexa-ethylene glycol linker. In thiscase, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA isgenerally formed by triple-helix bonds. Table 1 provides examples ofmodified RNAi agents of the invention.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the degradationactivities of cellular enzymes, such as, for example, withoutlimitation, certain nucleases. Techniques for inhibiting the degradationactivity of cellular enzymes against nucleic acids are known in the artincluding, but not limited to, 2′-amino modifications, 2′-amino sugarmodifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkylsugar modifications, uncharged backbone modifications, morpholinomodifications, 2′-O-methyl modifications, and phosphoramidate (see,e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxylgroup of the nucleotides on a dsRNA is replaced by a chemical group,generally by a 2′-amino or a 2′-methyl group. Also, at least onenucleotide may be modified to form a locked nucleotide. Such lockednucleotide contains a methylene bridge that connects the 2′-oxygen ofribose with the 4′-carbon of ribose. Oligonucleotides containing thelocked nucleotide are described in Koshkin, A. A., et al., Tetrahedron(1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998),39: 5401-5404). Introduction of a locked nucleotide into anoligonucleotide improves the affinity for complementary sequences andincreases the melting temperature by several degrees (Braasch, D. A. andD. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption aswell as targeting to a particular tissue or uptake by specific types ofcells such as liver cells. In certain instances, a hydrophobic ligand isconjugated to the dsRNA to facilitate direct permeation of the cellularmembrane and or uptake across the liver cells. Alternatively, the ligandconjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides as well as dsRNA agents. Forexample, cholesterol has been conjugated to various antisenseoligonucleotides resulting in compounds that are substantially moreactive compared to their non-conjugated analogs. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103. Otherlipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-mediatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Li and coworkers report that attachment of folic acid to the 3′-terminusof an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated tooligonucleotides include polyethylene glycols, carbohydrate clusters,cross-linking agents, porphyrin conjugates, delivery peptides and lipidssuch as cholesterol.

In certain instances, conjugation of a cationic ligand tooligonucleotides results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the invention via reaction of a selected serum-binding ligandwith a linking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, an dsRNA bearing an aralkylligand attached to the 3′-terminus of the dsRNA is prepared by firstcovalently attaching a monomer building block to a controlled-pore-glasssupport via a long-chain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbuilding-block bound to the solid support. The monomer building blockmay be a nucleoside or other organic compound that is compatible withsolid-phase synthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat.No. 5,212,295, drawn to monomers for the preparation of oligonucleotideshaving chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and5,541,307, drawn to oligonucleotides having modified backbones; U.S.Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and thepreparation thereof through reductive coupling; U.S. Pat. No. 5,457,191,drawn to modified nucleobases based on the 3-deazapurine ring system andmethods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summarylisting of some of the oligonucleotide modifications known in the art isfound at, for example, PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences of theinvention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligandmolecules may be conjugated to oligonucleotides at the 5′-position bythe use of a ligand-nucleoside phosphoramidite wherein the ligand islinked to the 5′-hydroxy group directly or indirectly via a linker. Suchligand-nucleoside phosphoramidites are typically used at the end of anautomated synthesis procedure to provide a ligand-conjugatedoligonucleotide bearing the ligand at the 5′-terminus.

Examples of modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Examples of modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate. The use of acholesterol conjugate is particularly preferred since such a moiety canincrease targeting liver cells cells, a site of HAMP expression.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski Ret al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of the HAMP gene, such as pathological processeswhich can be mediated by down regulating HAMP gene expression, such asanemia and other diseases associated with lowered iron levels. Suchpharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for deliveryto the liver via parenteral delivery.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of the HAMP gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A dosage of 5 mg dsRNA per kilogram bodyweight of recipient per day is sufficient to inhibit or suppressexpression of the HAMP gene and may be administered systemically to thepatient.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 microgram to 1 mg per kilogram body weight per day.The pharmaceutical composition may be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processeswhich can be mediated by down regulating HAMP gene expression. Suchmodels are used for in vivo testing of dsRNA, as well as for determininga therapeutically effective dose.

Any method can be used to administer a dsRNA of the present invention toa mammal. For example, administration can be direct; oral; or parenteral(e.g., by subcutaneous, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection), or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations).

Typically, when treating a mammal with anemia and other diseasesassociated with lowered iron levels, the dsRNA molecules areadministered systemically via parental means. For example, dsRNAs,conjugated or unconjugate or formulated with or without liposomes, canbe administered intravenously to a patient. For such, a dsRNA moleculecan be formulated into compositions such as sterile and non-sterileaqueous solutions, non-aqueous solutions in common solvents such asalcohols, or solutions in liquid or solid oil bases. Such solutions alsocan contain buffers, diluents, and other suitable additives. Forparenteral, intrathecal, or intraventricular administration, a dsRNAmolecule can be formulated into compositions such as sterile aqueoussolutions, which also can contain buffers, diluents, and other suitableadditives (e.g., penetration enhancers, carrier compounds, and otherpharmaceutically acceptable carriers).

In addition, dsRNA molecules can be administered to a mammal as biologicor abiologic means as described in, for example, U.S. Pat. No.6,271,359. Abiologic delivery can be accomplished by a variety ofmethods including, without limitation, (1) loading liposomes with adsRNA acid molecule provided herein and (2) complexing a dsRNA moleculewith lipids or liposomes to form nucleic acid-lipid or nucleicacid-liposome complexes. The liposome can be composed of cationic andneutral lipids commonly used to transfect cells in vitro. Cationiclipids can complex (e.g., charge-associate) with negatively chargednucleic acids to form liposomes. Examples of cationic liposomes include,without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP.Procedures for forming liposomes are well known in the art. Liposomecompositions can be formed, for example, from phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine.Numerous lipophilic agents are commercially available, includingLipofectin® (Invitrogen/Life Technologies, Carlsbad, Calif.) andEffectene™ (Qiagen, Valencia, Calif.). In addition, systemic deliverymethods can be optimized using commercially available cationic lipidssuch as DDAB or DOTAP, each of which can be mixed with a neutral lipidsuch as DOPE or cholesterol. In some cases, liposomes such as thosedescribed by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997))can be used. In other embodiments, polycations such as polyethyleneiminecan be used to achieve delivery in vivo and ex vivo (Boletta et al., J.Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding theuse of liposomes to deliver nucleic acids can be found in U.S. Pat. No.6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005.Nat. Biotechnol. 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including,without limitation, the use of viral vectors. For example, viral vectors(e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNAmolecules to liver cells. Standard molecular biology techniques can beused to introduce one or more of the dsRNAs provided herein into one ofthe many different viral vectors previously developed to deliver nucleicacid to cells. These resulting viral vectors can be used to deliver theone or more dsRNAs to cells by, for example, infection.

dsRNAs of the present invention can be formulated in a pharmaceuticallyacceptable carrier or diluent. A “pharmaceutically acceptable carrier”(also referred to herein as an “excipient”) is a pharmaceuticallyacceptable solvent, suspending agent, or any other pharmacologicallyinert vehicle. Pharmaceutically acceptable carriers can be liquid orsolid, and can be selected with the planned manner of administration inmind so as to provide for the desired bulk, consistency, and otherpertinent transport and chemical properties. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation:water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

In addition, dsRNA that target the HAMP gene can be formulated intocompositions containing the dsRNA admixed, encapsulated, conjugated, orotherwise associated with other molecules, molecular structures, ormixtures of nucleic acids. For example, a composition containing one ormore dsRNA agents that target the HAMP gene can contain othertherapeutic agents such as other lipid lowering agents (e.g., statins).

Methods for Treating Diseases that can be Modulated by Down Regulatingthe Expression of HAMP

The methods and compositions described herein can be used to treatdiseases and conditions that can be modulated by down regulating HAMPgene expression. For example, the compositions described herein can beused to treat anemia and other diseases associated with lowered ironlevels.

Methods for Inhibiting Expression of the HAMP Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the HAMP gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target HAMP gene is silenced. Because of their highspecificity, the dsRNAs of the invention specifically target RNAs(primary or processed) of the target HAMP gene. Compositions and methodsfor inhibiting the expression of these HAMP genes using dsRNAs can beperformed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of theHAMP gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol) administration. In preferred embodiments, thecompositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

Gene Walking of the HAMP Gene

Design and In Silico Selection of siRNAs Targeting Human Hepcidin

siRNAs targeting either human or mouse hepcidin antimicrobial peptide(also referred to as hepcidin, official symbol: hamp, Genbank accessionNM_(—)021175.2 (human) and NM_(—)032541.1 (mouse) were selectedaccording to following criteria:

a) predicted highest specificity in human or mouse or

b) cross-reactivity to cynomolgous monkey (macaca fascicularis), rhesusmonkey (macaca mulatta) and chimpanzee (pan troglodytes) and predictedhighest specificity of siRNA antisense strand in human

siRNAs with stretches of >=4 Gs in a row were excluded from theselection.

Specificity was predicted by fastA homology search algorithm andproprietary scripts and was defined as given, if every mRNA in the humanRefSeq database (release 17, dowloaded on May 9, 2006) except forhepcidin had either

-   -   a) at least 2 mismatches to the siRNA sense and antisense        sequence positions 10 to 18 (non-seed regions), with at least 1        mismatch in position 10 or 11 (cleavage site region) of the        respective strand if only 2 mismatches were present, or    -   b) at least 1 mismatch to the siRNA sense and antisense sequence        positions 2 to 9 (seed region)

Primate sequences were assembled from genomic sequences (available onJun. 8, 2006 at QFBase, Baylor College of Medicine and NCBI) previous tothe selection in order to obtain information on conserved regions withhuman hepcidin, which were defined as candidate target regions for theset of cross-reactive siRNAs.

Table 1 provides an identification of siRNAs designed to selectivelytarget the human hepcidin gene (with cross reactivity to orthologoushepcidin genes as described above).

Table 2 provides an identification of siRNAs designed to target themouse hepcidin gene.

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

dsRNA Expression Vectors

In another aspect of the invention, HAMP specific dsRNA molecules thatmodulate HAMP gene expression activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or Ul snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would beable to choose the appropriate regulatory/promoter sequence based on theintended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single HAMP gene or multiple HAMP genes over a period of a week ormore are also contemplated by the invention. Successful introduction ofthe vectors of the invention into host cells can be monitored usingvarious known methods. For example, transient transfection. can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection. of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The HAMP specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the instant disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

HAMP siRNA Screening COS-7 Cells

Cloning:

The cDNA sequences for human hepcidin and murine hepcidin-1 cDNA weresynthesized thereby introducing a 5′-XhoI- and a 3′-NotI site andsubcloned into pGA4 (Geneart AG, Regensburg, Germany). Human and mousehepcidin were subcloned via the introduced XhoI- and NotI-sites into themultiple cloning site of the psiCheck-2 vector (Promega, Mannheim,Germany), which is located downstream of the Renilla translational stopcodon. Correct subcloning was assured by sequencing (GATC Biotech,Konstanz, Germany).

Transfections:

Directly before plasmid transfection, Cos-7 cells (DSMZ, Braunschweig,Germany) were seeded at 1.5×10⁴ cells/well on 96-well plates (GreinerBio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium(Dulbecco's MEM, 10% fetal calf serum, 2 mM L-glutamine, 1.2 μg/mlsodium bicarbonate, 100 u penicillin/100 μg/ml streptomycin, all fromBiochrom AG, Berlin, Germany). 50 ng of plasmid/well were transfectedwith Lipofectamine-2000 (Invitrogen) as described below for the siRNAs,with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl/well,prepared as a mastermix for the whole plate.

4 h after the transfection of the plasmid, siRNA transfections wereperformed in quadruplicates. For each well 0.5 μl Lipofectamine-2000(Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM(Invitrogen) and incubated for 15 min at room temperature. For the siRNAconcentration being 50 nM in the 100 μl transfection volume, 1 μl of a 5μM siRNA were mixed with 10.5 μl Opti-MEM per well, combined with theLipofectamine-2000-Opti-MEM mixture and again incubated for 15 minutesat room temperature. During that incubation time, growth medium wasremoved from cells and replaced by 75 μl/well of fresh medium.siRNA-Lipofectamine-2000-complexes were applied completely (25 μl eachper well) to the cells and cells were incubated for 24 h at 37° C. and5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau, Germany).

Cells were harvested by lysis with the appropriate buffer from theDual-Glo Luciferase assay (Promega GmbH, Mannheim, Germany) and theassay was performed according to the kit's protocol. Values obtainedfrom the Renilla-luciferase measurement were normalized with therespective values acquired in the Firefly-luciferase measurement as atransfection and loading control. Values acquired with siRNAs directedagainst the Renilla-luciferase-hepcidin fusion mRNA were normalized tothe value obtained with an unspecific siRNA (directed against theneomycin resistance gene) which was set to 100%.

Effective siRNAs from the screen were further characterized by doseresponse curves. Transfections of dose response curves were performed in6-fold dilutions starting with 100 nM down to 10 fM. Mock (no siRNA) wasset to 100% expression level. siRNAs were diluted with Opti-MEM to afinal volume of 12.5 μl according to the above protocol. (FIGS. 3 and 4,Table 1)

As can be seen in FIGS. 3 and 4 (summarized in Table 1), many activedsRNAs to hepcidin are identified.

The above screening procedure was repeated using the murine hepcidingene as the target and the siRNAs of Table 2.

Stabilizing Modifications and Activity

Active duplexes identified above were then remade using modified basesand linkages in order to improve stability of the duplex and protect itfrom exo and endoribonuclease degradation. Table 3 (and Table 2 formurine selective siRNAs) provides a listing of the duplexes made and theactivities of these duplexes in the COS-7 assay described above. InTables 2 and 3, a lower case “s” represents a phosphorothioate linkageand a lower case base, e.g. “u”, represents a 2′OMe modified base, e.g.2′OMe-U.

Activity is provided from a 50 nM screen (duplicates) for human siRNA inTable 3 (Table 2 for murine) and shown in FIG. 5 (FIG. 6 for murine).Further, IC50 values were determined as described above for several ofthe most active agents. The results are provided in Table 3.

Activity of Murine Hepcidin siRNA In Vivo

Experimental Methods

The efficacy of AD-10812 was determined in normal 10 week old129s6/svEvTac mice using AD-1955 targeting luciferase as a control.These siRNAs were formulated in liposome (LNP-1) as described below andadministered through i.v. bolus at a dose of 10 mg/kg (n=8). Forty eighthours after injection, the liver and serum samples were harvested. Theliver Hamp1 and Hamp2 mRNA levels were determined by qRT-PCR using Hamp1and Hamp2 specific primers and serum iron levels were determined usingFeroxcine (Randox Life Sciences) and Hitachi 717 instrument.

Formulation of siRNAs in Liposomal Particles

The lipidoid LNP-01 4HCl (MW 1487) (FIG. 7), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used toprepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared: LNP-01, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-CeramideC16, 100 mg/mL. LNP-01, Cholesterol, and PEG-Ceramide C16 stocksolutions were then combined in a 42:48:10 molar ratio. Combined lipidsolution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5)such that the final ethanol concentration was 35-45% and the finalsodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticlesformed spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically>85%.

Results

Approximately 70% reduction in Hamp1 mRNA levels and 64% increase inserum iron levels were achieved 48 h after administration of AD-10812(FIG. 8: FIGS. 8A and 8B). AD-10812 did not reduce Hamp2 mRNA levels.

TABLE 1hepcidin siRNAs, double overhang design, sense strand: dTsdT; antisense strand: dTsdT% position in SEQ SEQ inhb duplex human ID ID (50 IC50 name access. #Sense sequence (5′-3′) NO Antisense sequence (5′-3′) NO nM) (nM) AD-9914378-396 CCCAGAACAUAGGUCUUGGTsT 1 CCAAGACCUAUGUUCUGGGTsT 37 78 AD-9915283-301 GCUGCUGUCAUCGAUCAAATsT 2 UUUGAUCGAUGACAGCAGCTsT 38 91 0.091AD-9916 154-172 CACAACAGACGGGACAACUTsT 3 AGUUGUCCCGUCUGUUGUGTsT 39 65AD-9917 56-74 CCAGACAGACGGCACGAUGTsT 4 CAUCGUGCCGUCUGUCUGGTsT 40 88 0.28AD-9918 312-330 UGCUGCAAGACGUAGAACCTsT 5 GGUUCUACGUCUUGCAGCATsT 41 72AD-9919 238-256 GAAGGAGGCGAGACACCCATsT 6 UGGGUGUCUCGCCUCCUUCTsT 42 840.2 AD-9920 315-333 UGCAAGACGUAGAACCUACTsT 7 GUAGGUUCUACGUCUUGCATsT 4385 0.025 AD-9921 158-176 ACAGACGGGACAACUUGCATsT 8 UGCAAGUUGUCCCGUCUGUTsT44 65 AD-9922 291-309 CAUCGAUCAAAGUGUGGGATsT 9 UCCCACACUUUGAUCGAUGTsT 4590 0.019 AD-9923 57-75 CAGACAGACGGCACGAUGGTsT 10 CCAUCGUGCCGUCUGUCUGTsT46 87 0.12 AD-9924 236-254 GCGAAGGAGGCGAGACACCTsT 11GGUGUCUCGCCUCCUUCGCTsT 47 68 AD-9925 243-261 AGGCGAGACACCCACUUCCTsT 12GGAAGUGGGUGUCUCGCCUTsT 48 82 0.18 AD-9926  4-22 UGUCACUCGGUCCCAGACATsT13 UGUCUGGGACCGAGUGACATsT 49 60 AD-9927 317-335 CAAGACGUAGAACCUACCUTsT14 AGGUAGGUUCUACGUCUUGTsT 50 74 AD-9928  6-24 UCACUCGGUCCCAGACACCTsT 15GGUGUCUGGGACCGAGUGATsT 51 7 AD-9929 153-171 CCACAACAGACGGGACAACTsT 16GUUGUCCCGUCUGUUGUGGTsT 52 68 AD-9930 156-174 CAACAGACGGGACAACUUGTsT 17CAAGUUGUCCCGUCUGUUGTsT 53 60 AD-9931 318-336 AAGACGUAGAACCUACCUGTsT 18CAGGUAGGUUCUACGUCUUTsT 54 69 AD-9932 225-243 AUGUUCCAGAGGCGAAGGATsT 19UCCUUCGCCUCUGGAACAUTsT 55 77 AD-9933 223-241 CCAUGUUCCAGAGGCGAAGTsT 20CUUCGCCUCUGGAACAUGGTsT 56 79 AD-9934 224-242 CAUGUUCCAGAGGCGAAGGTsT 21CCUUCGCCUCUGGAACAUGTsT 57 51 AD-9935 314-332 CUGCAAGACGUAGAACCUATsT 22UAGGUUCUACGUCUUGCAGTsT 58 89 0.11 AD-9936 321-339 ACGUAGAACCUACCUGCCCTsT23 GGGCAGGUAGGUUCUACGUTsT 59 51 AD-9937 288-306 UGUCAUCGAUCAAAGUGUGTsT24 CACACUUUGAUCGAUGACATsT 60 74 AD-9938 58-76 AGACAGACGGCACGAUGGCTsT 25GCCAUCGUGCCGUCUGUCUTsT 61 71 AD-9939 133-151 UGACCAGUGGCUCUGUUUUTsT 26AAAACAGAGCCACUGGUCATsT 62 58 AD-9940 65-83 CGGCACGAUGGCACUGAGCTsT 27GCUCAGUGCCAUCGUGCCGTsT 63 84 0.13 AD-9941 285-303 UGCUGUCAUCGAUCAAAGUTsT28 ACUUUGAUCGAUGACAGCATsT 64 88 0.061 AD-9942 382-400GAACAUAGGUCUUGGAAUATsT 29 UAUUCCAAGACCUAUGUUCTsT 65 90 0.016 AD-9943282-300 GGCUGCUGUCAUCGAUCAATsT 30 UUGAUCGAUGACAGCAGCCTsT 66 90 0.023AD-9944 284-302 CUGCUGUCAUCGAUCAAAGTsT 31 CUUUGAUCGAUGACAGCAGTsT 67 910.023 AD-9945 280-298 GCGGCUGCUGUCAUCGAUCTsT 32 GAUCGAUGACAGCAGCCGCTsT68 90 0.056 AD-9946 286-304 GCUGUCAUCGAUCAAAGUGTsT 33CACUUUGAUCGAUGACAGCTsT 69 84 0.11 AD-9947 287-305 CUGUCAUCGAUCAAAGUGUTsT34 ACACUUUGAUCGAUGACAGTsT 70 89 0.027 AD-9948 289-307GUCAUCGAUCAAAGUGUGGTsT 35 CCACACUUUGAUCGAUGACTsT 71 88 0.072 AD-9949155-173 ACAACAGACGGGACAACUUTsT 36 AAGUUGUCCCGUCUGUUGUTsT 72 62

TABLE 2Mouse cross reactive siRNAs: sequences and activity in COS-7 cellsTable 2A: Sequences position in mouse SEQ SEQ access.sense strand sequence ID antisense strand ID duplex # (5′-3′) NOsequence (5′-3′) NO name 171-189 CAGACAUUGCGAUACCAAUTsT 73AUUGGUAUCGCAAUGUCUGTsT 145 AD-9890 171-189 cAGAcAuuGcGAuAccAAuTsT 74AUUGGuAUCGcAAUGUCUGTsT 146 AD-10800 171-189 cAGAcAuuGcGAuAccAAuTsT 75AuuGGuAUCGcAAuGUCuGTsT 147 AD-10824 172-190 AGACAUUGCGAUACCAAUGTsT 76CAUUGGUAUCGCAAUGUCUTsT 148 AD-9891 172-190 AGAcAuuGcGAuAccAAuGTsT 77cAUUGGuAUCGcAAUGUCUTsT 149 AD-10801 172-190 AGAcAuuGcGAuAccAAuGTsT 78cAuuGGuAUCGcAAuGUCUTsT 150 AD-10825 170-188 GCAGACAUUGCGAUACCAATsT 79UUGGUAUCGCAAUGUCUGCTsT 151 AD-9892 170-188 GcAGAcAuuGcGAuAccAATsT 80UUGGuAUCGcAAUGUCUGCTsT 152 AD-10802 170-188 GcAGAcAuuGcGAuAccAATsT 81uuGGuAUCGcAAuGUCuGCTsT 153 AD-10826 284-302 UAGCCUAGAGCCACAUCCUTsT 82AGGAUGUGGCUCUAGGCUATsT 154 AD-9893 284-302 uAGccuAGAGccAcAuccuTsT 83AGGAUGUGGCUCuAGGCuATsT 155 AD-10803 284-302 uAGccuAGAGccAcAuccuTsT 84AGGAuGuGGCUCuAGGCuATsT 156 AD-10827 173-191 GACAUUGCGAUACCAAUGCTsT 85GCAUUGGUAUCGCAAUGUCTsT 157 AD-9894 173-191 GAcAuuGcGAuAccAAuGcTsT 86GcAUUGGuAUCGcAAUGUCTsT 158 AD-10804 173-191 GAcAuuGcGAuAccAAuGcTsT 87GcAuuGGuAUCGcAAuGUCTsT 159 AD-10828 177-195 UUGCGAUACCAAUGCAGAATsT 88UUCUGCAUUGGUAUCGCAATsT 160 AD-9895 177-195 uuGcGAuAccAAuGcAGAATsT 89UUCUGcAUUGGuAUCGcAATsT 161 AD-10805 177-195 uuGcGAuAccAAuGcAGAATsT 90uUCuGcAuuGGuAUCGcAATsT 162 AD-10829 178-196 UGCGAUACCAAUGCAGAAGTsT 91CUUCUGCAUUGGUAUCGCATsT 163 AD-9896 178-196 uGcGAuAccAAuGcAGAAGTsT 92CUUCUGcAUUGGuAUCGcATsT 164 AD-10806 178-196 uGcGAuAccAAuGcAGAAGTsT 93CuUCuGcAuuGGuAUCGcATsT 165 AD-10830 100-118 CACCACCUAUCUCCAUCAATsT 94UUGAUGGAGAUAGGUGGUGTsT 166 AD-9897 100-118 cAccAccuAucuccAucAATsT 95UUGAUGGAGAuAGGUGGUGTsT 167 AD-10807 100-118 cAccAccuAucuccAucAATsT 96uuGAuGGAGAuAGGuGGuGTsT 168 AD-10831 120-138 AGAUGAGACAGACUACAGATsT 97UCUGUAGUCUGUCUCAUCUTsT 169 AD-9898 120-138 AGAuGAGAcAGAcuAcAGATsT 98UCUGuAGUCUGUCUcAUCUTsT 170 AD-10808 120-138 AGAuGAGAcAGAcuAcAGATsT 99UCuGuAGUCuGUCUcAUCUTsT 171 AD-10832 176-194 AUUGCGAUACCAAUGCAGATsT 100UCUGCAUUGGUAUCGCAAUTsT 172 AD-9899 176-194 AuuGcGAuAccAAuGcAGATsT 101UCUGcAUUGGuAUCGcAAUTsT 173 AD-10809 176-194 AuuGcGAuAccAAuGcAGATsT 102UCuGcAuuGGuAUCGcAAUTsT 174 AD-10833 372-390 AAUAAAGACGAUUUUAUUUTsT 103AAAUAAAAUCGUCUUUAUUTsT 175 AD-9900 372-390 AAuAAAGAcGAuuuuAuuuTsT 104AAAuAAAAUCGUCUUuAUUTsT 176 AD-10810 372-390 AAuAAAGAcGAuuuuAuuuTsT 105AAAuAAAAUCGUCuuuAuUTsT 177 AD-10834 169-187 GGCAGACAUUGCGAUACCATsT 106UGGUAUCGCAAUGUCUGCCTsT 178 AD-9901 169-187 GGcAGAcAuuGcGAuAccATsT 107UGGuAUCGcAAUGUCUGCCTsT 179 AD-10811 169-187 GGcAGAcAuuGcGAuAccATsT 108uGGuAUCGcAAuGUCuGCCTsT 180 AD-10835 245-263 UGCUGUAACAAUUCCCAGUTsT 109ACUGGGAAUUGUUACAGCATsT 181 AD-9902 245-263 uGcuGuAAcAAuucccAGuTsT 110ACUGGGAAUUGUuAcAGcATsT 182 AD-10812 245-263 uGcuGuAAcAAuucccAGuTsT 111ACuGGGAAuuGuuAcAGcATsT 183 AD-10836 231-249 UCUUCUGCUGUAAAUGCUGTsT 112CAGCAUUUACAGCAGAAGATsT 184 AD-9903 231-249 ucuucuGcuGuAAAuGcuGTsT 113cAGcAUUuAcAGcAGAAGATsT 185 AD-10813 231-249 ucuucuGcuGuAAAuGcuGTsT 114cAGcAuuuAcAGcAGAAGATsT 186 AD-10837 60-78 CUGCCUGUCUCCUGCUUCUTsT 115AGAAGCAGGAGACAGGCAGTsT 187 AD-9904 60-78 cuGccuGucuccuGcuucuTsT 116AGAAGcAGGAGAcAGGcAGTsT 188 AD-10814 60-78 cuGccuGucuccuGcuucuTsT 117AGAAGcAGGAGAcAGGcAGTsT 189 61-79 UGCCUGUCUCCUGCUUCUCTsT 118GAGAAGCAGGAGACAGGCATsT 190 AD-9905 61-79 uGccuGucuccuGcuucucTsT 119GAGAAGcAGGAGAcAGGcATsT 191 AD-10815 61-79 uGccuGucuccuGcuucucTsT 120GAGAAGcAGGAGAcAGGcATsT 192 59-77 GCUGCCUGUCUCCUGCUUCTsT 121GAAGCAGGAGACAGGCAGCTsT 193 AD-9906 59-77 GcuGccuGucuccuGcuucTsT 122GAAGcAGGAGAcAGGcAGCTsT 194 AD-10816 59-77 GcuGccuGucuccuGcuucTsT 123GAAGcAGGAGAcAGGcAGCTsT 195 62-80 GCCUGUCUCCUGCUUCUCCTsT 124GGAGAAGCAGGAGACAGGCTsT 196 AD-9907 62-80 GccuGucuccuGcuucuccTsT 125GGAGAAGcAGGAGAcAGGCTsT 197 AD-10817 62-80 GccuGucuccuGcuucuccTsT 126GGAGAAGcAGGAGAcAGGCTsT 198 56-74 CAGGCUGCCUGUCUCCUGCTsT 127GCAGGAGACAGGCAGCCUGTsT 199 AD-9908 56-74 cAGGcuGccuGucuccuGcTsT 128GcAGGAGAcAGGcAGCCUGTsT 200 AD-10818 56-74 cAGGcuGccuGucuccuGcTsT 129GcAGGAGAcAGGcAGCCuGTsT 201 AD-10838 232-250 CUUCUGCUGUAAAUGCUGUTsT 130ACAGCAUUUACAGCAGAAGTsT 202 AD-9909 232-250 cuucuGcuGuAAAuGcuGuTsT 131AcAGcAUUuAcAGcAGAAGTsT 203 AD-10819 232-250 cuucuGcuGuAAAuGcuGuTsT 132AcAGcAuuuAcAGcAGAAGTsT 204 AD-10839 233-251 UUCUGCUGUAAAUGCUGUATsT 133UACAGCAUUUACAGCAGAATsT 205 AD-9910 233-251 uucuGcuGuAAAuGcuGuATsT 134uAcAGcAUUuAcAGcAGAATsT 206 AD-10820 233-251 uucuGcuGuAAAuGcuGuATsT 135uAcAGcAuuuAcAGcAGAATsT 207 AD-10840 234-252 UCUGCUGUAAAUGCUGUAATsT 136UUACAGCAUUUACAGCAGATsT 208 AD-9911 234-252 ucuGcuGuAAAuGcuGuAATsT 137UuAcAGcAUUuAcAGcAGATsT 209 AD-10821 234-252 ucuGcuGuAAAuGcuGuAATsT 138uuAcAGcAuuuAcAGcAGATsT 210 AD-10841 57-75 AGGCUGCCUGUCUCCUGCUTsT 139AGCAGGAGACAGGCAGCCUTsT 211 AD-9912 57-75 AGGcuGccuGucuccuGcuTsT 140AGcAGGAGAcAGGcAGCCUTsT 212 AD-10822 57-75 AGGcuGccuGucuccuGcuTsT 141AGcAGGAGAcAGGcAGcCUTsT 213 AD-10842 58-76 GGCUGCCUGUCUCCUGCUUTsT 142AAGCAGGAGACAGGCAGCCTsT 214 AD-9913 58-76 GGcuGccuGucuccuGcuuTsT 143AAGcAGGAGAcAGGcAGCCTsT 215 AD-10823 58-76 GGcuGccuGucuccuGcuuTsT 144AAGcAGGAGAcAGGcAGcCTsT 216 AD-10843 Table 2B. Activity. % position inhibin mouse at 50 access. duplex nM IC50 # name (%) (uM) 171-189 AD-9890 950.052 171-189 AD-10800 86 0.056 171-189 AD-10824 22 172-190 AD-9891 890.15 172-190 AD-10801 17 172-190 AD-10825 16 170-188 AD-9892 91 0.099170-188 AD-10802 23 170-188 AD-10826 44 284-302 AD-9893 70 284-302AD-10803 80 0.34 284-302 AD-10827 65 173-191 AD-9894 64 173-191 AD-1080427 173-191 AD-10828 0 177-195 AD-9895 71 177-195 AD-10805 40 177-195AD-10829 57 178-196 AD-9896 30 178-196 AD-10806 26 178-196 AD-10830 23100-118 AD-9897 62 100-118 AD-10807 11 100-118 AD-10831 3 120-138AD-9898 86 0.031 120-138 AD-10808 85 0.076 120-138 AD-10832 83 14176-194 AD-9899 61 176-194 AD-10809 62 176-194 AD-10833 61 372-390AD-9900 56 372-390 AD-10810 17 372-390 AD-10834 0 169-187 AD-9901 950.096 169-187 AD-10811 25 169-187 AD-10835 10 245-263 AD-9902 94 0.032245-263 AD-10812 92 0.03 245-263 AD-10836 88 0.065 231-249 AD-9903 69231-249 AD-10813 58 231-249 AD-10837 73 11 60-78 AD-9904 76 60-78AD-10814 29 60-78 61-79 AD-9905 43 61-79 AD-10815 18 61-79 59-77 AD-990671 59-77 AD-10816 2 59-77 62-80 AD-9907 72 62-80 AD-10817 3 62-80 56-74AD-9908 76 56-74 AD-10818 16 56-74 AD-10838 8 232-250 AD-9909 79 232-250AD-10819 39 232-250 AD-10839 35 233-251 AD-9910 70 233-251 AD-10820 55233-251 AD-10840 74 2.7 234-252 AD-9911 66 234-252 AD-10821 66 234-252AD-10841 58 57-75 AD-9912 56 57-75 AD-10822 0 57-75 AD-10842 0 58-76AD-9913 63 58-76 AD-10823 0 58-76 AD-10843 3

TABLE 3 Modified duplexes: sequences and activity in COS-7 cellsTable 3A: Sequences position in human SEQ SEQ access. parentSense strand sequence ID Antisense strand ID duplex # Duplex (5′-3′) NOsequence (5′-3′) NO name 283-301 AD-9915 GcuGcuGucAucGAucAAATsT 217uuuGAUCGAuGAcAGcAGCTsT 234 AD-11449 56-74 AD-9917 ccAGAcAGAcGGcAcGAuGTsT218 cAUCGuGCCGUCuGUCuGGTsT 235 AD-11450 238-256 AD-9919GAAGGAGGcGAGAcAcccATsT 219 uGGGuGUCUCGCCUCCuUCTsT 236 AD-11451 315-333AD-9920 uGcAAGAcGuAGAAccuAcTsT 220 GuAGGuUCuACGUCuUGcATsT 237 AD-11452291-309 AD-9922 cAucGAucAAAGuGuGGGATsT 221 UCCcAcACuuuGAUCGAuGTsT 238AD-11453 57-75 AD-9923 cAGAcAGAcGGcAcGAuGGTsT 222 CcAUCGuGCCGUCuGUCuGTsT239 AD-11454 243-261 AD-9925 AGGcGAGAcAcccAcuuccTsT 223GGAAGuGGGuGUCUCGCCUTsT 240 AD-11455 314-332 AD-9935cuGcAAGAcGuAGAAccuATsT 224 uAGGuUCuACGUCuuGcAGTsT 241 AD-11456 65-83AD-9940 cGGcAcGAuGGcAcuGAGcTsT 225 GCUcAGuGCcAUCGuGCCGTsT 242 AD-11457285-303 AD-9941 uGcuGucAucGAucAAAGuTsT 226 ACuuuGAUCGAuGAcAGcATsT 243AD-11458 382-400 AD-9942 GAAcAuAGGucuuGGAAuATsT 227uAuUCcAAGACCuAuGuUCTsT 244 AD-11459 282-300 AD-9943GGcuGcuGucAucGAucAATsT 228 uuGAUCGAuGAcAGcAGCCTsT 245 AD-11460 284-302AD-9944 cuGcuGucAucGAucAAAGTsT 229 CuuuGAUCGAuGAcAGcAGTsT 246 AD-11461280-298 AD-9945 GcGGcuGcuGucAucGAucTsT 230 GAUCGAuGAcAGcAGCCGCTsT 247AD-11462 286-304 AD-9946 GcuGucAucGAucAAAGuGTsT 231cACuuuGAUCGAuGAcAGCTsT 248 AD-11463 287-305 AD-9947cuGucAucGAucAAAGuGuTsT 232 AcACuuuGAUCGAuGAcAGTsT 249 AD-11464 289-307AD-9948 GucAucGAucAAAGuGuGGTsT 233 CcAcACuuuGAUCGAuGACTsT 250 AD-11465Table 3B: Activity position in human access. parent duplex % IC50 #Duplex name inhib (nM) 283-301 AD-9915 AD-11449 16 56-74 AD-9917AD-11450 84 4.04 238-256 AD-9919 AD-11451 10 315-333 AD-9920 AD-11452 60291-309 AD-9922 AD-11453 88 0.33 57-75 AD-9923 AD-11454 52 243-261AD-9925 AD-11455 37 314-332 AD-9935 AD-11456 63 65-83 AD-9940 AD-1145729 285-303 AD-9941 AD-11458 85 0.66 382-400 AD-9942 AD-11459 88 0.18282-300 AD-9943 AD-11460 21 284-302 AD-9944 AD-11461 28 280-298 AD-9945AD-11462 60 286-304 AD-9946 AD-11463 31 287-305 AD-9947 AD-11464 53289-307 AD-9948 AD-11465 55

We claim:
 1. A double-stranded ribonucleic acid (dsRNA), wherein thedsRNA comprises an antisense strand and a sense strand, wherein eachstrand is less than 30 nucleotides in length, and wherein the targetsequence of the dsRNA comprises the first nineteen nucleotides of SEQ IDNO:29 (GAACAUAGGUCUUGGAAUA).
 2. The dsRNA of claim 1, wherein the dsRNAcomprises at least one modified nucleotide.
 3. The dsRNA of claim 2,wherein the modified nucleotide is chosen from the group of: a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 4. ThedsRNA of claim 2, wherein the modified nucleotide is chosen from thegroup of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 5. ThedsRNA of claim 1, wherein the sense strand is modified as follows:GAAcAuAGGucuuGGAAuATsT (SEQ ID NO:227) and the antisense strand ismodified as follows: uAuUCcAAGACCuAuGuUCTs (SEQ ID NO:244), wherein “c”indicates a 2′-O-methyl modified cytodine; “u” indicates a 2′-O-methylmodified uracil, and sT indicates a 5′-phosphorothioate modifiedthymidine.
 6. The dsRNA of claim 1, wherein the sense strand consists ofSEQ ID NO:29 and the antisense strand consists of SEQ ID NO:65.
 7. Acell comprising the dsRNA of claim
 1. 8. The cell of claim 7 wherein thecell is a liver cell.
 9. A vector encoding each strand of the dsRNA ofclaim
 1. 10. A cell comprising the vector of claim
 9. 11. Apharmaceutical composition comprising the dsRNA of claim 1 and apharmaceutically acceptable carrier.
 12. The pharmaceutical compositionof claim 11, wherein the carrier is a lipid carrier.
 13. The dsRNA ofclaim 1, wherein the dsRNA is formulated in a lipid formulation.
 14. ThedsRNA of claim 1, wherein the dsRNA is formulated in a liposome.
 15. ThedsRNA of claim 1, wherein the dsRNA inhibits the expression of ahepcidin antimicrobial peptide (HAMP) gene in a COS-7 cell by 90%compared to a control when the dsRNA is transfected into the cell at aconcentration of 50 nM.
 16. The dsRNA of claim 5, wherein the dsRNAinhibits the expression of a hepcidin antimicrobial peptide (HAMP) genein a COS-7 cell by 88% compared to a control when the dsRNA istransfected into the cell at a concentration of 50 nM.
 17. The dsRNA ofclaim 1, whrerein the dsRNA is conjugated to a molecule.
 18. The dsRNAof claim 17, wherein the molecule is a ligand moiety or a non-ligandmoiety.
 19. A method for inhibiting the expression of a hepcidinantimicrobial peptide (HAMP) gene in a cell, the method comprising:introducing into the cell the dsRNA of claim 1; and maintaining the cellfor a time sufficient to obtain degradation of the mRNA transcript ofthe HAMP gene, thereby inhibiting expression of the HAMP gene in thecell.
 20. A method of treating or managing pathological processesmediated by hepcidin antimicrobial peptide (HAMP) expression in apatient in need thereof comprising administering to the patient aneffective amount of the dsRNA of claim 1.